Arterial stiffness is a result of normal aging, and it is also a significant risk factor for heart failure. While the changes in the large scale mechanics of blood flow caused by arterial stiffness are well-studied, the perturbations of the microenvironment of the endothelial cell layer are not as well studied. The purpose of this research plan is to look at endothelial monolayers on different stiffness substrates in order to mimic the changes in the endothelial microenvironment during disease. This will be done by investigating the cytoskeleton of the cells - particularly the vimentin intermediate filament network as it has been shown to play a role in permeability of the monolayer. The cytoskeleton of single cells and confluent monolayers will be characterized by immunofluorescence and Western blotting - including a method that will characterize the ratio of the insoluble and soluble vimentin fractions. The stiffness of single endothelial cells and the endothelial monolayer will be quantified with atomic force microscopy measurements. Changes in cell stiffness will also be measured after perturbing different cytoskeletal elements with drugs. Finally, the permeability of endothelial cell monolayers will be studied on different stiffness substrates by a novel assay based on penetration of fluorescently labeled particles into the underlying hydrogel substrate. This proposal will help to further understand the affect of stiffening the arteries on the endothelial monolayer.